Chromatographic stationary phase separation materials used in liquid chromatography are commonly based on a porous carrier of silica onto which a functionalisation has been made in order to achieve the desired separation characteristics for a certain analyte to be separated. Common types of functionalisation are the preparation of hydrophobic stationary phases, e.g. a C18-phase, in which usually octadecylsilane is used as a functionalising agent and reacted with silanol groups of porous silica. However, it is difficult to have all silanol groups reacting with the functionalising agent, mainly due to steric hindrance. Remaining silanol groups make the material very susceptible to hydrolysis, due to their polar nature. Therefore, end-capping of remaining silanol groups with, e.g. trimethylchlorosilane, is usually made. However, stationary phase separation materials based on silica, such as a conventional end-capped C18-phase, are still chemically rather unstable at high pH due to still remaining silanol groups, as well as to the fact that the end-capped silanol groups are still susceptible to hydrolysis.
The retention time and also capacity, the maximum amount of analyte applied without introducing competitive interaction, for a certain analyte depend on the actual surface coverage of the functionalisation. Moreover, the optimum surface coverage is a function of both the analyte and the actual surface functionalisation. The surface coverage obtained under normally applied procedures, which is a surface bonding on bare, fully rehydroxylated silica, is typically higher than optimum for all analytes.
A lowering of the surface coverage of functional groups on silica has according to one method according to the state of the art been achieved by, instead of using an excess, using a deficit of the functionalising agent, e.g. an organosilane. However, when using a deficit of functionalising agent the level of surface coverage is difficult to reproduce, due to, e.g., remaining moisture and exact determination of the chemically available surface. Also, hydrophobic organosilane molecules often tend to form clusters, which lead to “island” formation and thus an uneven distribution of the organosilane on the silica surface.
Furthermore, when using a deficit of the organosilane used for modification there will be remaining silanol groups present which are usually blocked with an end-capping agent such as a lower alkyl organo silane, e.g. trimethylchlorosilane. However, as already described, end-capped silanol groups are still susceptible to hydrolysis which means that the chemical stability will be even lower than, e.g., conventional C18-phases at extreme pH-values (<2 and >10). Also, end-capping is difficult to make complete which means that there will usually be remaining silanol groups present which are very susceptible to hydrolysis. Remaining silanol groups also interact unfavourably with analytes, especially basic analytes.
Alternatively, a lowering of the surface coverage of functional groups on silica can be made by end-capping some of the silanol groups prior to functionalisation as described in Marshall et al., J. of Chrom., 361 (1986) 71-92. Also in this case, there are problems with an even lower chemical stability than conventional C18-phases due to the increased number of end-capped silanol groups.
Several attempts have been made to provide stationary phase separation materials which are chemically stable. U.S. Pat. No. 4,017,528 A discloses preparation of a porous silicon dioxide bearing organic groups which are part both of the skeleton structure and the surface. U.S. Pat. No. 6,686,035 B2 discloses a porous inorganic/organic hybrid material particle comprising a polyorganoalkoxysiloxane through the whole particle. However, both these types of hybrid particles are much less mechanically stable than pure silica particles. US. 2005/0191503 A1 discloses a method of preparing a chemically stable separation material wherein a polycarbosilane layer is covalently bonded to a silica surface. U.S. Pat. No. 3,722,181 A discloses a polymeric stationary phase chemically bonded to a substrate of silica.
There is a need for chemically stable silica based stationary phase separation materials which are mechanically stable.
There is, furthermore, a need for a method of reducing the number of remaining silanol groups on a silica surface of a stationary phase separation material after functionalisation in order to increase the chemical stability of the stationary phase separation material and minimise unfavourable interaction with analytes.
There is also a need for a method of reducing the surface coverage of functional groups in a controlled way having high reproducibility.
There is also a need for a silica based stationary phase separation material wherein the number of remaining silanol groups on the silica surface is low, thereby increasing the stability of the stationary phase separation material and minimising unfavourable interaction with analytes.
There is also a need for a chemically stable silica based stationary phase material, which preserves all the performance benefits of bare silica functionalised with organosilanes, i.e., a common silica based functionalised stationary phase separation material.
There is therefore an object of the present invention to provide a silica based material which has high chemical and mechanical stability and onto which functionalisation can be made in a controlled way. There is also an object of the present invention to provide a stationary phase separation material for chromatography which has high chemical and mechanical stability, and which has high loadability. There is also an objective of the present invention to provide a stationary phase separation material which has all the performance benefits of state of the art surface functionalised silica based on pure silica at the same time the chemical stability is higher.